Figure 1.
The regulation of PTP opening by ubiquinone analogs depends on the cell lines.
The incubation medium contained 250 mM sucrose, 1 mM Pi-Tris, 10 mM Tris-MOPS, 5 mM succinate-Tris, 50 µM digitonin and 1 µM Calcium Green-5N. The final volume was 2 ml, pH 7.4, 25°C. Experiments were begun by the addition of 5.106 cells (isolated rat hepatocytes, MH1C1 or Clone-9) followed by the addition of ubiquinone 0 (Ub0), ubiquinone 5 (Ub5), ubiquinone 10 (Ub10) or decyl-ubiquinone (DUb) at the indicated concentrations. After 2 min of incubation, the Ca2+ Retention Capacity (CRC) was measured by adding Ca2+ pulses every 90 s until PTP opening. Results are mean ± S.D. of at least four independent experiments.
Figure 2.
Competition between PTP-inactive and PTP-active ubiquinone analogs on PTP regulation in Clone-9 and MH1C1 cells.
The Ca2+ Retention Capacity was measured as in Fig. 1. When indicated, 20 µM Ub0, 100 µM Ub5 or 100 µM Ub10 were added. The results represent the means ± S.D. of three independent experiments. *, p≤0.05; **, p≤0.01, unpaired Student's t test.
Figure 3.
Effect of ubiquinone analogs on MH1C1 and Clone-9 viability.
MH1C1 and Clone-9 cells were exposed for 30 min to serum-free culture medium supplemented or not with the indicated concentrations of Ub0 or Ub5 (left and right panels). When used in combination (middle panels), the concentrations were 20 µM for Ub0, 50 µM Ub5 and 100 µM for DUb. Cells were then incubated in normal medium for 24 h. The percentage of mortality represents the proportion of Annexin V-positive cells measured by flow cytometry. The results represent the means ± S.D of at least four independent experiments. **, p≤0.01; ***, p≤0.001, unpaired Student's t test. In preliminary experiments, we found that Annexin V-positive cells were mostly Propidium iodide positive.
Figure 4.
The effect of ubiquinone analogs on ROS production depends on the cell lines.
A - The incubation medium contained 250 mM sucrose, 1 mM Pi-Tris, 10 mM Tris-MOPS, and 5 µM H2DCFDA. The final volume was 2 ml, pH 7.4, 37°C. Experiments were begun by the addition of 5.106 cells (MH1C1 or Clone-9) followed by the addition of 20µM Ub0, 100µM Ub5, 100µM Ub10 or 100µM DUb. FUb/F0 represents the change in fluorescence during 5 min after the addition of ubiquinone analog divided by the change in fluorescence during 5 min before the addition of ubiquinone analog. Results are mean ± S.D. of at least three independent experiments. B - Clone-9 cells were exposed for 30 min to serum-free culture media supplemented or not with 20 µM Ub0, 200 µM tocopherol or 1 mM tiron. Cells were then incubated in normal medium for 24 h. The percentage of mortality represents the proportion of Annexin V-positive cells measured by flow cytometry. The results represent the means ± S.D of at least three independent experiments. *, p≤0.05; **, p≤0.01, unpaired Student's t test.
Figure 5.
Effect of Ub0 or Ub5 on coculture of MH1C1 plus Clone-9 cells.
Clone-9 cells labeled with PKH26 lipophilic dye were cocultured with MH1C1 cells for 24 h in complete F12K medium. Cocultures were exposed for 30 min to serum-free culture medium supplemented or not with 20 µM Ub0 or 100 µM Ub5. Cocultures were then incubated in complete F12K medium for 24 h. Annexin V-positive cells and PKH-26-positive cells were measured by flow cytometry. The indicated percentages represent the percentage of Annexin V-positive cells within each population in the shown experiment. Results are representative of six independent experiments.